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The invention relates to methods and compositions for forced evolution of a virus genome, such as a genome of an HIV-1 virus strain, to produce a variant virus having an altered phenotype that provides a desired property that may be advantageous for development of small animal models of viral diseases, and for the development of novel therapeutic approaches to viral diseases, among others (e.g., evolving a virus to replicate in an advantageous tissue culture system). The invention relates to novel viral genomes and virions which are capable of replication in non-human animals and cells, and further relates to transgenic non-human animals and cell lines capable of supporting replication of such evolved virus variants. The invention also relates to methods for identifying novel antiviral agents.
HIV-1 and AIDS
Human immunodeficiency virus type I (HIV-1) is a human retrovirus that is believed to be an etiologic agent of acquired immune deficiency syndrome (AIDS), an infectious disease characterized by a profound loss of immune system function. An aspect of HIV-1 disease is the typically delayed onset of disease symptoms, such as opportunistic infections, Kaposi""s sarcoma, dementia, and wasting syndrome. Often it may take 10 to 15 years after initial infection before symptoms are evident; however, in some instances disease onset is quite rapid. Moreover, the specific pathology of HIV-1 disease can be quite variable between individuals and between strains of the HIV-1 virus (for a review, see Field""s Virology, Third Edition, Fields et al. Eds., Lippincott-Raven Publishers, Vol. 2, Chapters 60 and 61). At present, HIV-1 appears to be almost always pathogenic in humans, and although certain chemotherapeutic agents (e.g., protease inhibitors, nucleoside analogs) have shown clinical promise in arresting or slowing HIV-1 disease, there is no established cure or preventative for HIV-1 disease at present.
Unfortunately, the HIV-1 virus is also characterized by an extraordinarily high frequency of mutational change, including deletions, base pair substitutions, insertions, and recombinations between HIV-1 genomes. It has been estimated that on the average at least one quarter of the progeny virus from a single cycle of retrovirus replication will have some kind of mutation relative to the parent genome, and recombination will further recombine these variant genomes (Temin H M (1989) Genome 31: 17). This characteristic of HIV-1 (and other lentiviruses, such as retroviruses) makes it difficult to obtain therapeutic solutions which the virus cannot escape due to its inherently high rate of mutation and propensity to generate variants which are resistant to the particular therapeutic solution selected. For this reason, the currently used therapeutic method to treat HIV-1 disease is to combine a cocktail of multiple chemotherapeutic agents (e.g., protease inhibitors and nucleoside analogs) to make it less likely that a resistant variant can arise during therapy. Nonetheless, it is almost certain that resistant HIV-1 variants will arise, particularly in view of imperfect patient compliance with chemotherapeutic regimens, pharmacogenetic differences between individuals in bioavailability of the chemotherapy agents, and use of partially degraded or inaccurately dispensed chemotherapy agents in less-advanced nations.
The globally circulating strains of HIV-1 exhibit extreme genetic diversity (Robertson et al. (1995) Nature 374: 124). To evaluate the extent of global HIV-1 variation, sequences of virus strains originating from numerous countries have been compared. These studies have shown that HIV-1 can be classified into two major groups, designated M and O, which are defined as distinct clusters on phylogenetic trees. Groups M comprises the great majority of HIV-1 isolates and can be further subdivided into at least nine sequence subtypes or clades, designated A to I, with additional variants being added to the classification scheme continually (Gao et al. (1996) J. Virol. 70: 1651). Given this degree of diversity, it is widely believed that a vaccine based on a single strain or subtype of HIV-1 will be unsuccessful against the larger spectrum of globally circulating HIV-1 variants, as well as against new variants which continually arise. Furthermore, the HIV-1 virus appears to undergo sequence variation and functional mutation in patients; isolates from different phases of HIV-1 infection exhibit stage-specific replication characteristics (Asjo et al. (1986) Lancet 2: 660; Cheng-Meyer et al. (1988) Science 240: 80; Fenyo et al. (1988) J. Virol. 62: 4414; Tersmette (1989) J. Virol. 63: 2118).
In view of the propensity of HIV-1 to undergo rapid mutation and generate variants that are resistant to chemotherapeutic agents and candidate xe2x80x9cuniversalxe2x80x9d vaccines, it is desirable to have non-human animal models of HV-1 replication and disease in order to speed the identification an d development of new generations of antiviral agents that can be used to treat resistant HIV-1 variants, or to prevent the generation of such variants in vivo. Unfortunately, such non-human models of HIV-1 disease are presently lacking.
Non-human Models of HIV-1 Disease
The absence of a suitable animal model has remained one of the major barriers to the development of an effective therapy for HIV-1 infection. Ideally, a readily available small animal model that could sustain HIV-1 infection and develop clinical symptoms that reflect the disease in humans would prove useful for modeling pathogenesis and developing new antiviral agents. An animal model that could duplicate human immune responses would greatly facilitate the development of vaccines. Unfortunately, no current model fulfills these varied needs (for review see, Klotman et al. (1995) AIDS 9: 313; Chang et al. (1996) Transfus. Sci. 17: 89; and Bonyhadi M L and Kaneshima H (June, 1997) Molec. Med. Today pp. 246-253; Mosier D E (September, 1996) Hosp. Prac. Pp. 41-60).
In general, non-human animals are not susceptible to infection with HIV-1 (Morrow et al. (1987) J. Gen. Virol. 68: 2253). However, several animal models exist in which to study retroviruses related to HIV-1 and their related pathology; these include SIV in macaque monkeys, FIV in cats, and murine acquired immunodeficiency syndrome virus (MAIDS) in mice, among others. HIV-1 replicates weakly in chimpanzees, but causes no detectable disease symptoms, and chimpanzees are quite expensive and not suited for large-scale studies. Lewis A D and Johnson P R (1995) TIBTECH 13: 142 discuss various non-human animal model systems and their limitations.
Several HIV-2 isolates, including three molecular clones of HIV-2 (HIV-2ROD, HIV-2SBL-ISY, and HIV-2UC1), have also been reported to infect macaques (M. mulatta and M. nemestrina) or baboons (Franchini, et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2433-2437; Barnett, et al. (1993) Journal of Virology 67, 1006-14; Boeri, et al. (1992) Journal of Virology 66, 4546-50; Castro, et al. (1991) Virology 184, 219-26; Franchini, et al. (1990) Journal of Virology 64, 4462-7; Putkonen, et al. (1990) Aids 4, 783-9; Putkonen, et al. (1991) Nature 352, 436-8).
As alternatives to the above, models of HIV-1 pathogenesis have been experimentally derived in mice that are transgenic for portions of the HIV genome or an entire HIV-1 genome, as well as in SCID mice which have been reconstituted with HIV-infected immune cells (Ramezani et al. (1996) Transfus. Sci. 17: 99; Chang et al. (1996) op.cit). HIV transgenic mice have been developed to model the in vivo regulation and pathological consequences of expression of various HIV open reading frames (ORFs), including known HIV structural genes; although some useful information might have been obtained from expression in various tissues of such animals, HIV gene expression in T cells of HIV transgenic mice has been negligible, indicating a substantial limitation of these mice as HIV disease models. Furthermore, a major hindrance of any mouse system is the inability of HIV to infect mouse cells, even when these are transduced with the gene for human CD4, the major receptor protein for HIV-1 infectivity.
SCID/hu mice have been reported as candidate animal models for studying HIV-1 (Mosier D E (1996) op.cit; Aldrovandi G M and Zack J A (1996) J. Virol. 70: 1505; Bonyhadi M L and Kaneshima H (1997) op.cit). SCID mice lack mature mouse T and B cells, and have been successfully engrafted with human hematolymphoid organs (e.g., the SCID/hu mouse having engrafted human thymus and liver tissue, peripheral blood leukocytes (PBLs), or hematopoietic precursor cells (Kamel-Reid et al. (1988) Science 242: 1706; McCune et al. (1988) Science 241: 1632; Mosier et al. Nature 335: 256). Such xenochimeric SCID/hu or SCID/hu-PBL mice have been used to study HIV pathogenesis in vivo and to evaluate anti-HIV drugs (Mosier et al. (1991) Science 251: 791; Mosier et al. (1995) Science 260: 689; McCune et al. (1990) Science 247; 564; Ruprecht et al. (1992) AIDS Res. Hum. Retroviruses 8: 997). However, these SCID mice models produced certain results which were anomalous, such as when infected with non-cytopathic macrophage-tropic (in humans) HIV isolates the mice underwent a rapid depletion of CD4+ cells, but when infected with cytopathic, T cell-tropic HIV isolates the CD4+ cells were not depleted, the exact opposite of what occurs in the human.
Thus, the art continues to search for improved models of HIV disease using small animal models and different (i.e., non-HIV) viruses. The absence of a suitable animal model has remained one of the major barriers to the development of an effective therapy for HIV-1 infection. It is apparent from the foregoing that a need exists in the art for an improved model of HIV-1 infection to further the development of anti-HIV therapies and prophylactic agents.
Significant improvements to and new opportunities for anti-HIV therapies and antiviral screening methods could be realized if better models of HIV-1 replication and pathogenesis were available. The present invention meets these and other needs and provides such improvements and opportunities.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention. All publications cited are incorporated herein by reference, whether specifically noted as such or not.
The present invention relates to methods for generating viral genotypes encoding at least one modified viral tropic phenotype, such as infectivity, virulence, and pathogenesis in a cell type, tissue, or host animal species (commonly host range; defined herein as a subset of viral tropism). The tropic phenotype modification can either permit or restrict viral infection, replication, and/or cytopathic effect in a predetermined cell type and/or host species (e.g., a non-human mammal). A basic format of the method, termed viral genome shuffling, in broad application, consists of: (1) contacting a cell strain, cell line, or non-human animal (or explanted organ therefrom), which does not naturally support substantial replication of an predetermined virus, with at least one initial infectious virion or replicable genome of said predetermined virus under replication conditions, (2) recovering a plurality of replicated genome copies of said predetermined virus, either as virions or as viral genomes in polynucleotide form, wherein some or all of the replicated genome copies comprise a mutation relative to the initial infectious virion or replicable genome, (3) recombining a plurality of said replicated genome copies, so as to shuffle the mutations, thereby generating a collection of recombined replicated genome copies, and (4) selecting or screening said collection of recombined replicated genome copies to obtain one or more replicable viral genome encoding at least one modified viral tropic phenotype. It is often desirable to perform at least one additional iterative cycle whereby the collection of recombined replicated genome copies is contacted with the cell strain, cell line, or non-human animal (or explanted organ therefrom) under replication conditions to produce second (or subsequent) round replicated copies having additional mutations, and to recover and shuffle, by recombination, said second (or subsequent) round replicated genome copies prior to the step of selecting or screening for genomes encoding a modified viral tropic phenotype. Typically, the recombination in step (3) is performed in vitro or by an in vivo recombination method which substantially does not occur naturally during replication of said viral genome. In certain variations, naturally occurring in vivo recombination mechanisms can be used in conjunction with a collection of preselected virus variants having a desired phenotypic property to be optimized further; in this way, a natural viral recombination mechanism can be combined with intelligent selection of variants in an iterative manner to produce optimized variants by xe2x80x9cforced evolutionxe2x80x9d, wherein the forced evolved virus variants are not expected to, nor are observed to, occur in nature, nor are predicted to occur at an appreciable frequency. The practitioner may further elect to supplement and/or the mutational drift by introducing mutated viral genomes, or portions thereof, into the pool of initial infectious virions (or replicable genomes) and/or into the plurality of replicated genome copies which are recombined. Mutational drift may also be supplemented by the use of mutagens (e.g., chemical mutagens or mutagenic irradiation), or by employing replication conditions which enhance the mutation rate of the virus.
The invention also provides for the viral genomes and infectious virions produced by the method of viral genome shuffling; the exact structures of said produced viral genomes and infectious virions are definable a priori only by reference to the method by which they are generated. Thus, the invention includes a viral genome, or plurality thereof, produced by the methods described herein. The shuffled viral genome(s) produced thereby are easily distinguishable from naturally occurring viral genomes by virtue of their atypical modified viral tropic phenotype(s) which is/are normally not present in the population of naturally occurring viral genomes.
In a variation of the basic method, one or more portions of the viral genome are separately optimized or improved for function in the predetermined cell type and/or host species as distinct genetic elements isolated from the remainder of the viral genome. The optimized or improved portions of the viral genome are then either introduced into the initial viral genome(s) for use in the method, or are shuffled in by recombination with the replicated genome copies recovered after a round of replication in the host cell or host animal. In a variation, the optimized or improved portions of the viral genome can be used in conjunction with one or more heterologous polynucleotide sequence(s), such as non-viral genes or replicons to confer a desired functional or structural property, such as transcriptional regulation or translational regulation, to the heterologous sequence(s). Optimized or improved portions of a virus genome often can be marketed as a commercial product, either alone or in combination with one or more heterologous sequences.
The invention also encompasses compositions of such shuffled viral genomes encoding at least one modified viral tropic phenotype. The compositions can include a plurality of species of shuffled viral genomes, or can represent a single purified viral genome species. Certain shuffled viral genomes encode variant viruses which possess detectable phenotypes that are not naturally occurring and which can be selected for; selected phenotypes often are characterized by desirable properties, such as modified host range as compared to wildtype virus, modified cell tropism as compared to wildtype virus, and modified immunogenicity, among other desirable properties.
The invention also encompasses screening assays and kits comprising a composition of such shuffled viral genome(s) and a cell type, tissue, or host animal species for which said shuffled viral genome(s) encode a modified viral tropism or drug resistance phenotype. In an aspect, the screening assay or kit further comprises a test agent, which is typically a small organic molecule such as a nucleoside analog or protease inhibitor with a molecular weight of less than 3,000 Daltons. In an aspect, the cell type or host animal is transgenic and expresses at least one human protein which confers, either alone or in conjunction with one or more additional human protein species, susceptibility of a cell to infection by and/or replication of said predetermined virus.
The invention provides screening assay methods for identifying and quantitating pharmacological properties of antiviral compounds. An exemplary screening assay format to identify agents that modify replication of a virus, said method comprises the steps of: (1) contacting, under suitable conditions, a test agent with a screening composition comprising: (i) one or more variants of said virus evolved by viral genome shuffling so as to replicate in cells or in a non-human animal, which cells or non-human animal does not naturally support replication of said virus, and (ii) said cells or non-human animal, and (2) determining whether said test agent modifies replication of said variant(s) in said cells or non-human animal. In an embodiment, the test agent inhibits virus replication, although it should be possible to screen for test agents which modify other aspects of viral replication, such as replication potentiators, immune modulators (for use with non-human animal systems), and agents which modify the virus genetic expression program (e.g, late gene inhibitors, latency modifiers, and the like).
Although the methods of the invention are believed to be suitable for use with substantially any virus type, including plant viruses, bacterial viruses, and animal viruses, it is described with particular reference to HIV-1 for illustrative purposes. It is believed that, with regard to animal viruses, the method will find particular use in developing shuffled virus genomes of pathogenic or oncogenic viruses for which present-day non-human animal models are insufficient or lacking entirely. HIV-1 and the related HIV disease is only one example of such suitable viruses and their pathologies.
With reference to HIV-1, the invention provides a method for producing, by viral genome shuffling, at least one HIV-1 variant which is capable of substantial replication in a non-human cell type. In an embodiment, the invention provides a method for generating one or more HIV-1 genome(s) which encode(s) the phenotype of permissive replication in mouse cells that express at least one human protein which confers, either alone or in conjunction with one or more additional human protein species, susceptibility of a mouse cell to infection by and/or replication of HIV-1. Examples of such human susceptibility proteins for HIV-1 infection include, but are not limited to hCD4, hCCR5. hCXCR4, and other accessory proteins identified in the art. Often, non-human primate homologs of these proteins can be substituted. In an aspect, the method employs a transgenic non-human cell or animal containing at least one expression cassette which encodes and expresses at least one human HIV-1 susceptibility protein. The viral genome shuffling method using these transgenic cells and/or animals as replication media produces shuffled HIV variants which have improved tropism for infection and/or replication of the transgenic non-human cells or animals. The shuffled HIV variants may be backcrossed (e.g., by recombination) to one or more HIV isolate(s), with concomitant selection for retention of the property of improved tropism for the transgenic cells or animals, thereby retaining the minimal mutations necessary for the desired tropic phenotype while xe2x80x9cnativizingxe2x80x9d the remainder of the viral genome to conform with the chosen HIV isolate(s). By the use of backcrossing, it is believed possible to generate, by use of the method of the invention, HIV variants substantially corresponding to essentially any HIV clinical isolate or sequence-related category thereof (e.g., group, clade, etc.), wherein the variants possess a desired phenotypic property not naturally associated with HIV; an example of such a phenotypic property can be the capacity for substantial replication in non-human cells and non-human organisms, such as for example mouse cells and transgenic mice.
In an aspect, the methods of the invention can be used to modify the immunogenic properties of a virus (i.e., the phenotype being selected for is an immunological property). For example, a virus (or collection of virus species) can be evolved to evade a host organism immune system, such as a human or mouse immune system. Also for example, a virus (or collection of virus species) can be evolved so as to mimic one or more immunologic stages of virus evolution in vivo; e.g., the viral dynamics of HIV-1 infection of a human patient is characterized by a continual natural evolution of certain immunodominant viral epitopes so as to naturally evade the human immune systemxe2x80x94the present invention can be used to generate HIV-1 variants which mimic one or more later immunological stages of HIV infection; such variants may serve as candidate HIV-1 vaccines, among other uses.
In an aspect, the methods of the invention can be used to modify the metabolic properties of a virus (i.e., the phenotype being selected for is a resistance to one or more chemotherapeutic agent). For example, a virus (or collection of virus species) can be evolved to rapidly model the natural development of drug resistance to anti-HIV drugs. The present invention can be used to generate HIV-1 variants which are drug resistant; such variants can be used in screening assays to identify alternative chemotherapeutic agents to which the HIV variants are not cross-resistant, among other uses.
The disclosed method for altering a viral phenotype by iterative genome shuffling and phenotype selection is a pioneering method which enables a broad range of novel and advantageous viral and non-viral compositions, therapeutic and prophylactic methods and compositions, and apparatus which will be apparent to those skilled in the art in view of the present disclosure.